Viable tissue repair implants and methods of use

ABSTRACT

Biocompatible tissue implants are provided for repairing a tissue injury or defect. The tissue implants comprise a biological tissue slice that serves as a source of viable cells capable of tissue regeneration and/or repair. The biological tissue slice can be harvested from healthy tissue to have a geometry that is suitable for implantation at the site of the injury or defect. The harvested tissue slice is dimensioned to allow the viable cells contained within the tissue slice to migrate out and proliferate and integrate with tissue surrounding the injury or defect site. Methods for repairing a tissue injury or defect using the tissue implants are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

Not applicable.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

Not applicable.

FIELD OF THE INVENTION

The present invention relates to methods and apparatus for the treatmentof tissue injuries or defects. Specifically, the present inventionrelates to tissue repair and augmentation implants, and moreparticularly, to tissue implants having viable cells capable of tissueregeneration and integration with tissue surrounding the area to berepaired, as well as methods for using such tissue implants.

BACKGROUND OF THE INVENTION

Injuries to tissue, such as cartilage, skin, muscle, bone, tendon, andligament where the tissue has been injured or traumatized frequentlyrequire surgical intervention to repair the damage and facilitatehealing. Such surgical repairs can include suturing or otherwiserepairing the damaged tissue with known medical devices, augmenting thedamaged tissue with other tissue, using an implant, a graft or anycombination of these techniques. Despite these conventional methods oftissue repair, there continues to be a need for surgical solutions thatfacilitate the regeneration of new, healthy tissue to provide morereliable repair and healing of the injured or damaged tissue over thelong term.

The search for a reliable source of viable cells for tissue regenerationhas been pursued for years. Recent tissue engineering techniques forrepairing tissue have typically involved replacing or reconstructingdamaged or injured tissue with cells that have been manipulated ex vivoto stimulate new tissue growth. The cells are usually incorporated intoa delivery vehicle (e.g., a scaffold or surgical implant) for placementat the tissue site, whereupon new tissue can be grown. Various surgicalimplants are known and have been used in surgical procedures to helpachieve these benefits. For example, it is known to use various devicesand techniques for creating implants having isolated cells loaded onto adelivery vehicle. Such cell-seeded implants have been used in an invitro method of making and/or repairing cartilage by growingcartilaginous structures that consist of chondrocytes seeded ontobiodegradable, biocompatible fibrous polymeric matrices as well asmatrices developed from collagenous materials. Such methods require theinitial isolation of chondrocytes from cartilaginous tissue prior to thechondrocytes being seeded onto the polymeric matrices. Other techniquesfor repairing damaged tissue employ implants having stem or progenitorcells that are used to produce the desired tissue. For example, it isknown to use stem or progenitor cells, such as the cells within fattytissue, muscle, bone marrow, or embryonic tissue to regenerate bone,cartilage, and other soft tissues in a patient. For example, stem cellsfrom fat are removed from the patient and placed in an environmentfavorable to cartilage formation, thereby inducing the cells toproliferate and to create a different type of cell, such as cartilagecells.

While the trend towards using tissue engineering approaches to tissuerepair continues to gain popularity, mainly because of the long-termbenefits provided to the patient, these current techniques are notwithout drawbacks. One disadvantage with current tissue engineeringtechniques is that they can be time consuming. A typical processinvolves the harvest of a tissue sample from the patient in a firstsurgical procedure, which is then transported to a laboratory for cellisolation, culture and amplification. The cell sample is grown for aperiod of 3 to 4 weeks using standard cell culture techniques to createa cell bank. Once the cell population has reached a target number, thecells are sent back to the surgeon for implantation during a secondsurgical procedure. This manual, labor-intensive process is extremelycostly and time consuming. Although the clinical data suggest long-termbenefits for the patient, the prohibitive cost of the procedure,combined with the traumatic impact of two surgical procedures, hashampered adoption of these techniques.

One method for tissue repair has been to place into a defect site animplant that is composed of cultured and amplified cells and a scaffold,which provides structural integrity and a surface area for cell adhesionand proliferation. In the past, such scaffolds have consisted mostly oftwo- or three-dimensional porous scaffolds that allow cell invasion andremodeling once the scaffold has been combined with living cells and hasbeen delivered inside the patient. This model is limited in applicationbecause of the secondary surgery and high costs involved. And thoughallografts have been used for tissue repair in the past, this solutionis also not ideal because of the limited availability of graft materialand the potential for disease transmission.

For these reasons, there continues to exist a need in this art for noveldevices and methods for regenerating tissue which are less timeconsuming and easier to implement. It is also desirable to provide animplant which can serve as a reliable source of viable cells, and whichcan be made in a quick and efficient manner for immediate use duringsurgery. There is thus a need for a less costly solution to repairingtissue defects or injuries that also provides the advantages of tissueregeneration, without the encumbrances of the currently availabledevices and methods of tissue repair previously mentioned.

SUMMARY OF THE INVENTION

The present invention provides a biocompatible tissue implant forrepairing a tissue defect or injury which comprises a biological tissueslice that serves as a source of viable cells capable of tissueregeneration and/or repair. The biological tissue slice can be harvestedfrom healthy tissue during the tissue repair surgery to have a geometrythat is suitable for implantation at the site of the injury or defect.The harvested tissue slice is dimensioned to allow the viable cellscontained within the tissue slice to migrate out and proliferate andintegrate with tissue surrounding the tissue repair site. The implantcan be delivered to the tissue site either alone or with a retainingelement to secure the implant to the injury or defect site. In oneembodiment, the harvested tissue slice can be combined with mincedtissue fragments to further enhance tissue regrowth. The minced tissuefragments can be delivered in a hydrogel or adhesive, which can alsofunction as the retaining element. Optionally, a biologically activeagent can be added to the implant at the tissue repair site to furtherenhance tissue healing or regeneration.

In another embodiment of the present invention, the implant can comprisemore than one tissue slice. The plurality of tissue slices can be joinedtogether to form a layered tissue implant having a desired size andgeometry suitable for implantation at the injury or defect site. In yetanother embodiment, a tissue slice can be joined to a tissue scaffold toform a composite implant. The implant can comprise a plurality of bothtissue slices and scaffold layers. The scaffold can further include abiologically active agent that enhances the effectiveness of the viablecells contained within the tissue slice to grow and integrate with thesurrounding tissue area.

The present invention also provides a method of treating injured ordiseased tissue using the biocompatible tissue implants of the presentinvention that involves delivering the tissue implant to the site of thetissue injury or defect. The tissue implant can optionally be secured tothe tissue site with a retaining element. Once implanted, the viablecells contained within the implant can begin regenerating new tissue tobe integrated into the tissue surrounding the repair site. Thebiocompatible tissue implants of the present invention can be used forthe repair and/or regeneration of diseased or damaged tissue. Further,the tissue implants can be used for tissue bulking, cosmetic treatments,therapeutic treatments, tissue augmentation, and tissue remodeling. Inembodiments in which the implant is used for tissue repair, the tissuerepair implant can be used to treat a variety of injuries, such as forexample, injuries occurring within the musculoskeletal system, such asrotator cuff injuries, anterior cruciate ligament (ACL) ruptures, ormeniscal tears, as well as injuries occurring in other connectivetissues, such as skin and cartilage. Furthermore, such implants can beused in other orthopaedic surgical procedures, such as hand and footsurgery, to repair tissues such as ligaments, nerves, and tendons.

By harvesting the tissue slice from viable, healthy tissue during thetissue repair surgery, the present invention provides a cell source forrepairing the tissue injury or defect at minimal cost and without theneed for additional surgeries. This method allows for the delivery ofviable cells to an injury or defect site without the cost of cellisolation and amplification. Further, because the present invention doesnot require the tissue slice to be minced to fine particles,manipulation time is reduced and the viability of the cells within thetissue is improved. An additional advantage of using a tissue slice as acell source for viable, healthy cells is that the tissue slice canprovide a native tissue surface for the biocompatible tissue implant,which will then have similar mechanical properties to that ofneighboring tissue. The tissue slice also provides a structure forbetter retention of the cells at the injury or defect site that can beeasily fixed to the site using conventional methods such as sutures,staples, or glues. In addition, by using a thin tissue slice, the cellshave the ability to migrate out from the tissue and provide goodintegration between the implanted tissue and the injury or defect site.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention can be more fully understood from the following detaileddescription taken in conjunction with the accompanying exemplarydrawing, in which:

FIG. 1A illustrates an exemplary embodiment of the tissue implantsecured to a tissue defect with a retaining element of the presentinvention;

FIG. 1B illustrates the tissue implant of FIG. 1A secured to a tissuedefect with another retaining element of the present invention;

FIG. 1C illustrates the tissue implant of FIG. 1A secured to a tissuedefect with yet another retaining element of the present invention;

FIG. 2A illustrates another exemplary embodiment of the tissue implantsecured to a tissue defect with a retaining element of the presentinvention;

FIG. 2B illustrates the tissue implant of FIG. 2A secured to a tissuedefect with another retaining element of the present invention;

FIG. 2C illustrates the tissue implant of FIG. 2A secured to a tissuedefect with yet another retaining element of the present invention;

FIG. 3A illustrates yet another exemplary embodiment of the tissueimplant secured to a tissue defect with a retaining element of thepresent invention;

FIG. 3B illustrates the tissue implant of FIG. 3A secured to a tissuedefect with another retaining element of the present invention;

FIG. 3C illustrates the tissue implant of FIG. 3A secured to a tissuedefect with yet another retaining element of the present invention;

FIG. 4A illustrates the tissue implant of FIG. 3A secured to anothertissue defect;

FIG. 4B illustrates the tissue implant of FIG. 4A with an additionalretaining element;

FIG. 5 represents a bar chart comparing DNA content between shreddedbovine ACL tissue and minced bovine ACL tissue fragments seeded onto atissue scaffold in vitro, at 4 days and 21 days.

FIGS. 6A-6C are photomicrographs of histological sections of samplesobtained after 3 weeks following the procedure of EXAMPLE 2,demonstrating cell migration from a meniscal tissue sample into apolymer scaffold;

FIG. 7A is a photograph of a cartilage sample obtained following theprocedure of EXAMPLE 3, demonstrating that minced cartilage fragmentscombined with cartilage tissue plugs enhance cell migration in spacesbetween the fragments and the plugs;

FIG. 7B is a photograph of a cartilage sample obtained following theprocedure of EXAMPLE 3, demonstrating that cartilage plugs culturedtogether as a bundle, without minced cartilage tissue fragments, did notbond together; and

FIG. 7C is a photomicrograph of a histological section of the sample ofFIG. 7A, demonstrating cell migration in the space between the mincedcartilage fragments and the cartilage plugs.

DETAILED DESCRIPTION OF THE INVENTION

The biocompatible tissue implants of the present invention are used inthe treatment of various types of tissue for various purposes. Forexample, the implants can be used for the repair and/or regeneration ofdiseased or damaged tissue, or they can be used for tissue bulking,tissue augmentation, cosmetic treatments, therapeutic treatments, andfor tissue sealing. The tissue implants include a tissue slice or stripharvested from healthy tissue that contains viable cells capable oftissue regeneration and/or remodeling. The tissue slice is harvested tohave a geometry that is suitable for implantation at the site of theinjury or defect. The harvested tissue slice is dimensioned to allow theviable cells contained within the tissue slice to migrate out andproliferate and integrate with tissue surrounding the repair site.

Although the implants are sometimes referred to herein as “tissue repairimplants” and the methods of using the implants are sometimescharacterized as tissue repair techniques, it is understood that theimplants can be used for a variety of tissue treatments, including butnot limited to tissue repair, tissue bulking, cosmetic treatments,therapeutic treatments, tissue remodeling or augmentation, and tissuesealing.

The term “viable,” as used herein, refers to a tissue sample having oneor more viable cells. Virtually any type of tissue can be used toconstruct the tissue repair implants of the present invention.Preferably, the tissue used is selected from cartilage tissue, meniscaltissue, ligament tissue, tendon tissue, skin tissue, bone tissue, muscletissue, periosteal tissue, pericardial tissue, synovial tissue, nervetissue, fat tissue, kidney tissue, bone marrow, liver tissue, bladdertissue, pancreas tissue, spleen tissue, intervertebral disc tissue,embryonic tissue, periodontal tissue, vascular tissue, blood andcombinations thereof. In one embodiment useful for cartilage repair, thetissue is free of bone tissue and is selected from the group consistingof cartilage tissue, meniscal tissue, periosteal tissue, fat tissue,bone marrow, blood, synovial tissue, ligament tissue and tendon tissue.The tissue used to construct the tissue implant can be autogeneictissue, allogeneic tissue, or xenogeneic tissue.

The term “slice,” as used herein, refers to a thin section, strip orsliver derived from any of the tissue types described above and used toconstruct the tissue implant. Preferably, the tissue slice has athickness less than about 1 mm, and more preferably has a thickness inthe range of about 200 μm to about 500 μm. A thin profile ensures propermigration of the cells out of the tissue slice. It is understood,however, that the tissue slice can have any length or width appropriatefor implantation at the defect, since these parameters do not greatlyaffect cell migration out of the tissue slice.

In one aspect of the invention, the tissue slices can be combined withfinely minced tissue fragments to enhance the effectiveness of theregrowth and healing response. In such an embodiment, the tissue slicescan be as thick as about 3 mm. However, the tissue slices are preferablybetween about 200 μm to about 1 mm.

In another aspect of the invention, the sliced tissue may be contactedwith a matrix-digesting enzyme to facilitate cell migration out of theextracellular matrix surrounding the cells. The enzymes can be used toincrease the rate of cell migration out of the extracellular matrix andinto the tissue defect or injury, or scaffold material. Suitablematrix-digesting enzymes that can be used in the present inventioninclude, but are not limited to, collagenase, chondroitinase, trypsin,elastase, hyaluronidase, peptidase, thermolysin, matrixmetalloproteinase, gelatinase and protease.

In one embodiment useful for meniscal repair, the tissue used in thetissue repair implant can be selected from the group consisting ofmeniscal tissue, cartilage tissue, skin, synovial tissue, periostealtissue, pericardial tissue, fat tissue, bone marrow, blood, tendontissue, ligament tissue, or combinations thereof. In one embodimentuseful for ligament repair, the tissue used in the tissue repair implantcan be selected from the group consisting of tendon tissue, ligamenttissue of the same type that is to be repaired, ligament tissue of adifferent type than the tissue that is to be repaired, synovial tissue,periosteal tissue, fascia, skin, and combinations thereof.

Turning now to the drawings and particularly to FIG. 1A, an exemplaryembodiment of the biocompatible tissue implant 20 of the presentinvention is shown. In the illustrated example, the tissue implant 20 isused to repair a cartilage defect 10. The tissue implant 20 comprises atissue slice 22 that has been harvested from healthy, viable cartilagetissue to have a geometry that is suitable for implantation at thedefect 10. The tissue slice 22 serves as a source of viable cartilagecells for repairing the cartilage defect, and is dimensioned to allowthe viable cells contained within the tissue slice 22 to migrate out andproliferate and integrate with the cartilage tissue 12 surrounding thedefect 10. To ensure proper migration of the cells out of the tissueimplant 20, the tissue slice 22 has a thickness less than about 1 mm.Preferably, the tissue slice 22 has a thickness in the range of about200 μm to about 500 μm, and can have any length or width appropriate forimplantation at the defect 10.

The tissue implant 20 can be delivered to the cartilage defect 10 andretained at the site of implantation by the force of compression againstthe tissue implant 20 by the surrounding cartilage tissue 12. Forinstance, the tissue implant 20 can be dimensioned to have a slightlylarger overall size than the area of the defect so that, uponimplantation, the tissue implant 20 can form a tight, interference fitwithin the defect 10. Alternatively, as illustrated in FIGS. 1A through1C, the tissue implant 20 can be secured using any conventional methodsuch as with a retaining element 30 to fix the tissue implant 20 to thedefect 10. The retaining element 30 can comprise a fastener, staple,tissue tack, suture, adhesive, or any combination of these. One skilledin the art will appreciate that the retaining element 30 is not limited,however, to such examples, and can comprise other suitable tissueattachment devices known in the art. Further, a number of factors candetermine which retaining element 30 is selected, including the size ofthe defect, the type of tissue being repaired, and the availability andcost of the retaining element 30.

FIG. 1A illustrates the tissue implant 20 secured in place with a staple32 which anchors to bone tissue 14 around the cartilage defect 10. Thetissue implant 20 can also be secured in place with an adhesive 34 asshown in FIG. 1B. Suitable adhesives 34 include, but are not limited to,hyaluronic acid, fibrin glue, fibrin clot, collagen gel, collagen-basedadhesive, alginate gel, crosslinked alginate,gelatin-resorcin-formalin-based adhesive, mussel-based adhesive,dihydroxyphenylalanine (DOPA)-based adhesive, chitosan,transglutaminase, poly(amino acid)-based adhesive, cellulose-basedadhesive, polysaccharide-based adhesive, synthetic acrylate-basedadhesives, platelet rich plasma (PRP), platelet poor plasma (PPP), PRPclot, PPP clot, blood, blood clot, blood component, blood componentclot, polyethylene glycol-based adhesive, Matrigel, MonostearoylGlycerol co-Succinate (MGSA), Monostearoyl Glycerolco-Succinate/polyethylene glycol (MGSA/PEG) copolymers, laminin,elastin, proteoglycans, and combinations thereof. As shown in FIG. 1C,the tissue implant 20 can also be fixed in place using sutures 36.

The tissue implant 20 can also be used in conjunction with minced tissueto enhance tissue repair. For example, minced tissue fragments can beadded to the adhesive 34 to further improve the tissue regenerationand/or remodeling process. Alternatively, the minced tissue fragmentscan be delivered in a gel-like carrier which is applied to the tissueimplant 20 at the defect 10. The minced tissue fragments can fill in thespaces between the tissue slice 22 and the defect 10. In such anembodiment in which minced tissue fragments are combined with the tissueslice, the thickness of the tissue slice forming the tissue implant 20can be about 3 mm, but preferably is between about 200 μm and about 1mm. By way of non-limiting example, the gel-like carrier can be abiological or synthetic hydrogel such as hyaluronic acid, fibrin glue,fibrin clot, collagen gel, collagen-based adhesive, alginate gel,crosslinked alginate, chitosan, synthetic acrylate-based gels, plateletrich plasma (PRP), platelet poor plasma (PPP), PRP clot, PPP clot,blood, blood clot, blood component, blood component clot, Matrigel,agarose, chitin, chitosan, polysaccharides, poly(oxyalkylene), acopolymer of poly(ethylene oxide)-poly(propylene oxide), poly(vinylalcohol), laminin, elasti, proteoglycans, solubilized basement membrane,or combinations thereof.

The minced tissue fragments can be obtained using any of a variety ofconventional techniques, such as for example, by biopsy or othersurgical removal. Preferably, the tissue sample is obtained during therepair surgery to minimize the total number of surgeries performed onthe patient. Once a sample of living tissue has been obtained, thesample can then be processed under sterile conditions to create asuspension having at least one minced, or finely divided, tissueparticle. It is also possible to harvest the tissue in minced form suchthat further processing is not necessary. The particle size of eachtissue fragment can vary, for example, the tissue size can be in therange of about 0.1 and 3 mm³, in the range of about 0.5 and 1 mm³, inthe range of about 1 to 2 mm³, or in the range of about 2 to 3 mm³, butpreferably the tissue particle is less than 1 mm³.

Preferably, the minced tissue has at least one viable cell that canmigrate from the tissue fragment. More preferably, the tissue containsan effective amount of cells that can migrate from the tissue fragmentand begin populating the tissue surrounding the defect 10. In anoptional embodiment, the minced tissue fragments may be contacted with amatrix-digesting enzyme to facilitate cell migration out of theextracellular matrix surrounding the cells. The enzymes are used toincrease the rate of cell migration out of the extracellular matrix andinto the scaffold material. Suitable matrix-digesting enzymes that canbe used in the present invention include, but are not limited to,collagenase, chondroitinase, trypsin, elastase, hyaluronidase,peptidase, thermolysin, matrix metalloproteinase, gelatinase andprotease. Preferably, the concentration of minced tissue particles inthe gel-carrier is in the range of approximately 1 to 1000 mg/cm³, andmore preferably in the range of about 1 to 200 mg/cm³.

While it is understood that a single tissue slice 22 is sufficient toform the tissue implant 20 of the present invention, the same principlesof cell migration and integration also apply to a layered tissue implant40 comprising a plurality of tissue slices 22. As illustrated in FIGS.2A through 2C, a plurality of tissue slices 22 can be joined together toform a layered tissue implant 40 of the present invention. The term“joined,” as used herein, broadly refers to the process of combiningtissue slices together, such as by the placement of a layer of tissueonto another layer of tissue, either alone or with an additionalretaining or adhesive element. Each of the tissue slices 22 can beuniformly sized, or they can be differently sized to form a layeredimplant 40 having an overall geometry and dimensions suitable forimplantation at the site of injury 10. Likewise, the number of tissueslices 22 to be joined together also depends upon the size of thedefect, and the size of each of the slices 22. However, to ensure propermigration of the cells out of the tissue implant 40, each of the tissueslice 22 should have a thickness less than about 1 mm as previouslydescribed. Preferably, each of the tissue slices 22 has a thickness inthe range of about 200 μm to about 500 μm.

Similar to the tissue implant 20 described above, the layered implant 40can be placed at the tissue defect 10 either alone, or with a retainingelement 30 as previously mentioned. In FIG. 2A, the tissue implant 40 issecured to a cartilage defect 10 using a staple 32 that anchors theimplant 40 to bone tissue 14 near the defect 10. In FIG. 2B, the tissueimplant 40 is held in place with a adhesive 34 such as the ones listedabove. To further enhance tissue regeneration and/or remodeling, mincedtissue fragments can be mixed in with the adhesive. In such anembodiment in which minced tissue fragments are combined with the tissueslices, the thickness of each tissue slice forming the layered implant40 can be about 3 mm, but preferably is between about 200 μm and about 1mm. Finally, the tissue implant 40 can be secured to the cartilagetissue 12 surrounding the defect 10 with sutures 36. After the layeredtissue implant 40 has been delivered to the defect 10, tissue regrowthcan be further enhanced by applying minced tissue fragments in agel-like carrier to the tissue implant 40 to fill in the spaces betweenthe tissue slices 22.

In yet another embodiment of the present invention, the tissue slice 22can be combined with a tissue scaffold 52 to form a composite tissueimplant 50 as illustrated in FIGS. 3A through 3C. For example, thetissue slice 22 can be placed on the tissue scaffold 52 and delivered tothe defect 10 as a composite implant 50. The composite tissue implant 50can be secured to the cartilage defect 10 using a retaining element 30such as a staple 32 as shown in FIG. 3A. Alternatively, as illustratedin FIG. 3B the composite tissue implant 50 can be fixed in place usingan adhesive 34 such as the ones described above, or using sutures 36 asshown in FIG. 3C. To further enhance tissue regeneration and/orremodeling, minced tissue fragments can be mixed in with the adhesive.In addition, minced tissue fragments in a gel-like carrier can beapplied to fill the spaces between the tissue slice 22, tissue scaffold52, and the defect 10 to enhance tissue growth.

Although illustrated as having a single tissue slice 22 and a singletissue scaffold 52, it is envisioned that the composite tissue implant50 of the present invention can include a plurality of layers of eithertissue slices 22 or tissue scaffolds 52. For instance, in one embodimenta plurality of tissue slices 22 can be sandwiched between layers of thetissue scaffold 52 to form a multilayered, composite implant 50. Inanother embodiment, the tissue slices 22 and tissue scaffolds 52 can bealternately layered onto one another to form the multilayered, compositeimplant 50. One skilled in the art will recognize that the number andorientation of tissue slices 22 and scaffolds 52 in the compositeimplant 50 can vary depending on the size of the defect 10, the type oftissue to be repaired, and the availability of the materials.

With the present embodiment, the tissue scaffold 52 can offer severaladvantages to the composite implant 50. A tissue scaffold 52 providesadditional structural integrity for cellular growth to occur. The tissuescaffold 52 also provides structural support for the tissue slice 22itself, which can be necessary to help retain the implant 50 in placefor certain tissue repairs. For example, in a partial meniscalreplacement shown in FIG. 4A, the tissue scaffold 52 provides additionalstrength to the tissue slice 22 of the composite tissue implant 50 sothat the implant 50 can be secured by sutures 36 to the meniscal tissue.If necessary or desired, a combination of retaining elements 30 can beused to secure the composite implant 50 to the meniscal tissue 60. Asshown in FIG. 4B, the composite implant 50 can be secured using bothsutures 36 and an adhesive or glue 34. Another advantage provided bytissue scaffolds is that they can act as a delivery vehicle forbioactive agents or effectors which enhance the overall effectiveness ofthe viable cells to grow and integrate with the tissue surrounding thedefect 10.

It is contemplated that the tissue scaffold 52 can be formed usingvirtually any material or delivery vehicle that is biocompatible andthat has sufficient structural integrity and physical and/or mechanicalproperties to effectively provide for ease of handling in an operatingroom environment. Sufficient strength and physical properties aredeveloped in the scaffold through the selection of materials used toform the scaffold, and the manufacturing process. In some embodiments,the scaffold is also pliable so as to allow the scaffold to adjust tothe dimensions of the target site of implantation. For instance, thescaffold can comprise a gel-like material or an adhesive material, aswell as a foam or mesh structure. Preferably, the scaffold can be abioresorbable or bioabsorbable material.

In one embodiment of the present invention, the scaffold can be formedfrom a biocompatible polymer. A variety of biocompatible polymers can beused to make the biocompatible tissue implants or scaffold devicesaccording to the present invention. The biocompatible polymers can besynthetic polymers, natural polymers or combinations thereof. As usedherein the term “synthetic polymer” refers to polymers that are notfound in nature, even if the polymers are made from naturally occurringbiomaterials. The term “natural polymer” refers to polymers that arenaturally occurring. In embodiments where the scaffold includes at leastone synthetic polymer, suitable biocompatible synthetic polymers caninclude polymers selected from the group consisting of aliphaticpolyesters, poly(amino acids), copoly(ether-esters), polyalkylenesoxalates, polyamides, tyrosine derived polycarbonates,poly(iminocarbonates), polyorthoesters, polyoxaesters, polyamidoesters,polyoxaesters containing amine groups, poly(anhydrides),polyphosphazenes, poly(propylene fumarate), polyurethane, poly(esterurethane), poly(ether urethane), and blends and copolymers thereof.Suitable synthetic polymers for use in the present invention can alsoinclude biosynthetic polymers based on sequences found in collagen,laminin, glycosaminoglycans, elastin, thrombin, fibronectin, starches,poly(amino acid), gelatin, alginate, pectin, fibrin, oxidized cellulose,chitin, chitosan, tropoelastin, hyaluronic acid, silk, ribonucleicacids, deoxyribonucleic acids, polypeptides, proteins, polysaccharides,polynucleotides and combinations thereof.

For the purpose of this invention aliphatic polyesters include, but arenot limited to, homopolymers and copolymers of lactide (which includeslactic acid, D-,L- and meso lactide); glycolide (including glycolicacid); ε-caprolactone; p-dioxanone (1,4-dioxan-2-one); trimethylenecarbonate (1,3-dioxan-2-one); alkyl derivatives of trimethylenecarbonate; δ-valerolactone; β-butyrolactone; γ-butyrolactone;ε-decalactone; hydroxybutyrate; hydroxyvalerate; 1,4-dioxepan-2-one(including its dimer 1,5,8,12-tetraoxacyclotetradecane-7,14-dione);1,5-dioxepan-2-one; 6,6-dimethyl-1,4-dioxan-2-one; 2,5-diketomorpholine;pivalolactone; α,αdiethylpropiolactone; ethylene carbonate; ethyleneoxalate; 3-methyl-1,4-dioxane-2,5-dione;3,3-diethyl-1,4-dioxan-2,5-dione; 6,6-dimethyl-dioxepan-2-one;6,8-dioxabicycloctane-7-one and polymer blends thereof. Aliphaticpolyesters used in the present invention can be homopolymers orcopolymers (random, block, segmented, tapered blocks, graft, triblock,etc.) having a linear, branched or star structure. Other useful polymersinclude polyphosphazenes, co-, ter- and higher order mixed monomer basedpolymers made from L-lactide, D,L-lactide, lactic acid, glycolide,glycolic acid, para-dioxanone, trimethylene carbonate andε-caprolactone.

As used herein, the term “glycolide” is understood to includepolyglycolic acid. Further, the term “lactide” is understood to includeL-lactide, D-lactide, blends thereof, and lactic acid polymers andcopolymers.

Elastomeric copolymers are also particularly useful in the presentinvention. Suitable elastomeric polymers include those with an inherentviscosity in the range of about 1.2 dL/g to 4 dL/g, more preferablyabout 1.2 dL/g to 2 dL/g and most preferably about 1.4 dL/g to 2 dL/g asdetermined at 25° C. in a 0.1 gram per deciliter (g/dL) solution ofpolymer in hexafluoroisopropanol (HFIP). Further, suitable elastomersexhibit a high percent elongation and a low modulus, while possessinggood tensile strength and good recovery characteristics. In thepreferred embodiments of this invention, the elastomer exhibits apercent elongation greater than about 200 percent and preferably greaterthan about 500 percent. In addition to these elongation and modulusproperties, suitable elastomers should also have a tensile strengthgreater than about 500 psi, preferably greater than about 1,000 psi, anda tear strength of greater than about 50 lbs/inch, preferably greaterthan about 80 lbs/inch.

Exemplary biocompatible elastomers that can be used in the presentinvention include, but are not limited to, elastomeric copolymers ofε-caprolactone and glycolide with a mole ratio of ε-caprolactone toglycolide of from about 35:65 to about 65:35, more preferably from 45:55to 35:65; elastomeric copolymers of ε-caprolactone and lactide(including L-lactide, D-lactide, blends thereof, and lactic acidpolymers and copolymers) where the mole ratio of ε-caprolactone tolactide is from about 95:5 to about 30:70 and more preferably from 45:55to 30:70 or from about 95:5 to about 85:15; elastomeric copolymers ofp-dioxanone (1,4-dioxan-2-one) and lactide (including L-lactide,D-lactide, blends thereof, and lactic acid polymers and copolymers)where the mole ratio of p-dioxanone to lactide is from about 40:60 toabout 60:40; elastomeric copolymers of ε-caprolactone and p-dioxanonewhere the mole ratio of ε-caprolactone to p-dioxanone is from about from30:70 to about 70:30; elastomeric copolymers of p-dioxanone andtrimethylene carbonate where the mole ratio of p-dioxanone totrimethylene carbonate is from about 30:70 to about 70:30; elastomericcopolymers of trimethylene carbonate and glycolide (includingpolyglycolic acid) where the mole ratio of trimethylene carbonate toglycolide is from about 30:70 to about 70:30; elastomeric copolymers oftrimethylene carbonate and lactide (including L-lactide, D-lactide,blends thereof, and lactic acid polymers and copolymers) where the moleratio of trimethylene carbonate to lactide is from about 30:70 to about70:30; and blends thereof. Examples of suitable biocompatible elastomersare described in U.S. Pat. No. 5,468,253.

In one embodiment, the elastomer is a copolymer of 35:65 ε-caprolactoneand glycolide, formed in a dioxane solvent and including a polydioxanonemesh. In another embodiment, the elastomer is a copolymer of 40:60ε-caprolactone and lactide with a polydioxanone mesh. In yet anotherembodiment, the elastomer is a 50:50 blend of a 35:65 copolymer ofε-caprolactone and glycolide and 40:60 copolymer of ε-caprolactone andlactide. The polydioxanone mesh may be in the form of a one layer thicktwo-dimensional mesh or a multi-layer thick three-dimensional mesh.

The scaffold of the present invention can, optionally, be formed from abioresorbable or bioabsorbable material that has the ability to resorbin a timely fashion in the body environment. The differences in theabsorption time under in vivo conditions can also be the basis forcombining two different copolymers when forming the scaffolds of thepresent invention. For example, a copolymer of 35:65 ε-caprolactone andglycolide (a relatively fast absorbing polymer) can be blended with40:60 ε-caprolactone and L-lactide copolymer (a relatively slowabsorbing polymer) to form a biocompatible scaffold. Depending upon theprocessing technique used, the two constituents can be either randomlyinter-connected bicontinuous phases, or the constituents could have agradient-like architecture in the form of a laminate type composite witha well integrated interface between the two constituent layers. Themicrostructure of these scaffolds can be optimized to regenerate orrepair the desired anatomical features of the tissue that is beingregrown.

In one embodiment, it is desirable to use polymer blends to formscaffolds which transition from one composition to another compositionin a gradient-like architecture. Scaffolds having this gradient-likearchitecture are particularly advantageous in tissue engineeringapplications to repair or regenerate the structure of naturallyoccurring tissue such as cartilage (articular, meniscal, septal,tracheal, auricular, costal, etc.), tendon, ligament, nerve, esophagus,skin, bone, and vascular tissue. For example, by blending an elastomerof ε-caprolactone-co-glycolide with ε-caprolactone-co-lactide (e.g.,with a mole ratio of about 5:95) a scaffold may be formed thattransitions from a softer spongy material to a stiffer more rigidmaterial, for example, in a manner similar to the transition fromcartilage to bone. Clearly, one skilled in the art will appreciate thatother polymer blends may be used for similar gradient effects, or toprovide different gradients (e.g., different absorption profiles, stressresponse profiles, or different degrees of elasticity).

The biocompatible scaffold 52 of the tissue repair implant 50 of thepresent invention can also include a reinforcing material comprised ofany absorbable or non-absorbable textile having, for example, woven,knitted, warped knitted (i.e., lace-like), non-woven, and braidedstructures. In one embodiment, the reinforcing material has a mesh-likestructure. In any of the above structures, mechanical properties of thematerial can be altered by changing the density or texture of thematerial, the type of knit or weave of the material, the thickness ofthe material, or by embedding particles in the material. The mechanicalproperties of the material may also be altered by creating sites withinthe mesh where the fibers are physically bonded with each other orphysically bonded with another agent, such as, for example, an adhesiveor a polymer. The fibers used to make the reinforcing component can bemonofilaments, yarns, threads, braids, or bundles of fibers. Thesefibers can be made of any biocompatible material including bioabsorbablematerials such as polylactic acid (PLA), polyglycolic acid (PGA),polycaprolactone (PCL), polydioxanone (PDO), trimethylene carbonate(TMC), copolymers or blends thereof. These fibers can also be made fromany biocompatible materials based on natural polymers including silk andcollagen-based materials. These fibers can also be made of anybiocompatible fiber that is nonresorbable, such as, for example,polyethylene, polyethylene terephthalate, poly(tetrafluoroethylene),polycarbonate, polypropylene and poly(vinyl alcohol). In one embodiment,the fibers are formed from 95:5 copolymer of lactide and glycolide.

In another embodiment, the fibers that form the reinforcing material canbe made of a bioabsorbable glass. Bioglass, a silicate containingcalcium phosphate glass, or calcium phosphate glass with varying amountsof solid particles added to control resorption time are examples ofmaterials that could be spun into glass fibers and used for thereinforcing material. Suitable solid particles that may be added includeiron, magnesium, sodium, potassium, and combinations thereof.

The biocompatible scaffolds as well as the reinforcing material may alsobe formed from a thin, perforation-containing elastomeric sheet withpores or perforations to allow tissue ingrowth. Such a sheet could bemade of blends or copolymers of polylactic acid (PLA), polyglycolic acid(PGA), polycaprolactone (PCL), and polydioxanone (PDO).

In one embodiment, filaments that form the biocompatible scaffolds 52 orthe reinforcing material may be co-extruded to produce a filament with asheath/core construction. Such filaments are comprised of a sheath ofbiodegradable polymer that surrounds one or more cores comprised ofanother biodegradable polymer. Filaments with a fast-absorbing sheathsurrounding a slower-absorbing core may be desirable in instances whereextended support is necessary for tissue ingrowth.

One skilled in the art will appreciate that one or more layers of thereinforcing material may be used to reinforce the tissue implant of theinvention. In addition, biodegradable textile scaffolds, such as, forexample, meshes, of the same structure and chemistry or differentstructures and chemistries can be overlaid on top of one another tofabricate biocompatible tissue implants with superior mechanicalstrength.

In embodiments where the scaffold includes at least one natural polymer,suitable examples of natural polymers include, but are not limited to,fibrin-based materials, collagen-based materials, hyaluronic acid-basedmaterials, glycoprotein-based materials, cellulose-based materials,silks and combinations thereof. By way of nonlimiting example, thebiocompatible scaffold can be constructed from a collagen-based smallintestine submucosa.

By way of non-limiting example, the scaffolds 52 of the presentinvention can be highly porous to allow cell growth therein. Preferably,the median pore size is in the range of about 100 to 500 microns. Inthese embodiments, the scaffold should be sufficiently pliable toaccommodate tissue growth within the interior region of the scaffold, sothat the geometry of the scaffold can be remodeled as tissue ingrowthincreases. Accordingly, in the present invention, tissue can be grown onthe surface of the biocompatible scaffold, or alternatively, tissue canbe grown into and on the surface of the biocompatible scaffold, suchthat the tissue becomes embedded in and integrated with the scaffold.

In another embodiment of the present invention, the biocompatiblescaffold 52 can be formed from a biocompatible ceramic material.Suitable biocompatible ceramic materials include, for example,hydroxyapatite, α-tricalcium phosphate, β-tricalcium phosphate,bioactive glass, calcium phosphate, calcium sulfate, calcium carbonate,xenogeneic and allogeneic bone material and combinations thereof.Suitable bioactive glass materials for use in the present inventioninclude silicates containing calcium phosphate glass, or calciumphosphate glass with varying amounts of solid particles added to controlresorption time. Suitable compounds that may be incorporated into thecalcium phosphate bioactive glass include, but are not limited to,magnesium oxide, sodium oxide, potassium oxide, and combinationsthereof.

In yet another embodiment of the tissue implants of the presentinvention, the scaffold 52 can be formed using tissue grafts, such asmay be obtained from autogeneic tissue, allogeneic tissue and xenogeneictissue. By way of non-limiting example, tissues such as skin, cartilage,ligament, tendon, periosteum, perichondrium, synovium, fascia, mesenterand sinew can be used as tissue grafts to form the biocompatiblescaffold 52. In some embodiments where an allogeneic tissue is used,tissue from a fetus or newborns can be used to avoid the immunogenicityassociated with some adult tissues.

In still yet another embodiment of the tissue implants, the scaffold canbe formed from a polymeric foam component having pores with an open cellpore structure. The pore size can vary, but preferably, the pores aresized to allow tissue ingrowth. More preferably, the pore size is in therange of about 50 to 1000 microns, and even more preferably, in therange of about 50 to 500 microns. The polymeric foam component can,optionally, contain a reinforcing component, such as for example, thetextiles disclosed above. In some embodiments where the polymeric foamcomponent contains a reinforcing component, the foam component can beintegrated with the reinforcing component such that the pores of thefoam component penetrate the mesh of the reinforcing component andinterlock with the reinforcing component.

The foam component of the tissue implant may be formed as a foam by avariety of techniques well known to those having ordinary skill in theart. For example, the polymeric starting materials may be foamed bylyophilization, supercritical solvent foaming (i.e., as described in EP464,163), gas injection extrusion, gas injection molding or casting withan extractable material (e.g., salts, sugar or similar suitablematerials).

In one embodiment, the foam component of the tissue repair implants ofthe present invention may be made by a polymer-solvent phase separationtechnique, such as lyophilization. Generally, however, a polymersolution can be separated into two phases by any one of the fourtechniques: (a) thermally induced gelation/crystallization; (b)non-solvent induced separation of solvent and polymer phases; (c)chemically induced phase separation, and (d) thermally induced spinodaldecomposition. The polymer solution is separated in a controlled mannerinto either two distinct phases or two bicontinuous phases. Subsequentremoval of the solvent phase usually leaves a porous structure with adensity less than the bulk polymer and pores in the micrometer rangesresulting in a porous polymer structure or an interconnected open cellporous foam. See Microcellular Foams Via Phase Separation, J. Vac. Sci.Technol., A. T. Young, Vol. 4(3), May/June 1986.

The applicable polymer concentration or amount of solvent that may beutilized will vary with each system. Generally, the amount of polymer inthe solution can vary from about 0.5% to about 90% and, preferably, willvary from about 0.5% to about 30% by weight, depending on factors suchas the solubility of the polymer in a given solvent and the finalproperties desired in the foam.

In one embodiment, solids may be added to the polymer-solvent system tomodify the composition of the resulting foam surfaces. As the addedparticles settle out of solution to the bottom surface, regions will becreated that will have the composition of the added solids, not thefoamed polymeric material. Alternatively, the added solids may be moreconcentrated in desired regions (i.e., near the top, sides, or bottom)of the resulting tissue implant, thus causing compositional changes inall such regions. For example, concentration of solids in selectedlocations can be accomplished by adding metallic solids to a solutionplaced in a mold made of a magnetic material (or vice versa).

A variety of types of solids can be added to the polymer-solvent system.Preferably, the solids are of a type that will not react with thepolymer or the solvent. Generally, the added solids have an averagediameter of less than about 1.0 mm and preferably will have an averagediameter of about 50 to about 500 microns. Preferably, the solids arepresent in an amount such that they will constitute from about 1 toabout 50 volume percent of the total volume of the particle andpolymer-solvent mixture (wherein the total volume percent equals 100volume percent).

Exemplary solids include, but are not limited to, particles ofdemineralized bone, calcium phosphate particles, bioglass particles,calcium sulfate, or calcium carbonate particles for bone repair,leachable solids for pore creation and particles of bioabsorbablepolymers not soluble in the solvent system that are effective asreinforcing materials or to create pores as they are absorbed, andnon-bioabsorbable materials.

Suitable leachable solids include nontoxic leachable materials such assalts (e.g., sodium chloride, potassium chloride, calcium chloride,sodium tartrate, sodium citrate, and the like), biocompatible mono anddisaccharides (e.g., glucose, fructose, dextrose, maltose, lactose andsucrose), polysaccharides (e.g., starch, alginate, chitosan), watersoluble proteins (e.g., gelatin and agarose). The leachable materialscan be removed by immersing the foam with the leachable material in asolvent in which the particle is soluble for a sufficient amount of timeto allow leaching of substantially all of the particles, but which doesnot dissolve or detrimentally alter the foam. The preferred extractionsolvent is water, most preferably distilled-deionized water. Such aprocess is described in U.S. Pat. No. 5,514,378. Preferably the foamwill be dried after the leaching process is complete at low temperatureand/or vacuum to minimize hydrolysis of the foam unless acceleratedabsorption of the foam is desired.

Suitable non-bioabsorbable materials include biocompatible metals suchas stainless steel, cobalt chrome, titanium and titanium alloys, andbioinert ceramic particles (e.g., alumina, zirconia, and calcium sulfateparticles). Further, the non-bioabsorbable materials may includepolymers such as polyethylene, polyvinylacetate, polymethylmethacrylate,polypropylene, poly(ethylene terephthalate), silicone, polyethyleneoxide, polyethylene glycol, polyurethanes, polyvinyl alcohol, naturalpolymers (e.g., cellulose particles, chitin, and keratin), andfluorinated polymers and copolymers (e.g., polyvinylidene fluoride,polytetrafluoroethylene, and hexafluoropropylene).

It is also possible to add solids (e.g., barium sulfate) that willrender the tissue implants radio opaque. The solids that may be addedalso include those that will promote tissue regeneration or regrowth, aswell as those that act as buffers, reinforcing materials or porositymodifiers.

As noted above, porous, reinforced tissue repair implant devices of thepresent invention are made by injecting, pouring, or otherwise placing,the appropriate polymer solution into a mold set-up comprised of a moldand the reinforcing elements of the present invention. The mold set-upis cooled in an appropriate bath or on a refrigerated shelf and thenlyophilized, thereby providing a reinforced scaffold. A bioactive agentcan be added either before or after the lyophilization step. In thecourse of forming the foam component, it is believed to be important tocontrol the rate of freezing of the polymer-solvent system. The type ofpore morphology that is developed during the freezing step is a functionof factors such as the solution thermodynamics, freezing rate,temperature to which it is cooled, concentration of the solution, andwhether homogeneous or heterogenous nucleation occurs. One of ordinaryskill in the art can readily optimize the parameters without undueexperimentation.

The required general processing steps include the selection of theappropriate materials from which the polymeric foam and the reinforcingcomponents are made. If a mesh reinforcing material is used, the propermesh density must be selected. Further, the reinforcing material must beproperly aligned in the mold, the polymer solution must be added at anappropriate rate and, preferably, into a mold that is tilted at anappropriate angle to avoid the formation of air bubbles, and the polymersolution must be lyophilized.

In embodiments that utilize a mesh reinforcing material, the reinforcingmesh has to be of a certain density. That is, the openings in the meshmaterial must be sufficiently small to render the construct sutureableor otherwise fastenable, but not so small as to impede proper bondingbetween the foam and the reinforcing mesh as the foam material and theopen cells and cell walls thereof penetrate the mesh openings. Withoutproper bonding the integrity of the layered structure is compromisedleaving the construct fragile and difficult to handle. Because thedensity of the mesh determines the mechanical strength of the construct,the density of the mesh can vary according to the desired use for tissuerepair. In addition, the type of weave used in the mesh can determinethe directionality of the mechanical strength of the construct, as wellas the mechanical properties of the reinforcing material, such as forexample, the elasticity, stiffness, burst strength, suture retentionstrength and ultimate tensile strength of the construct. By way ofnon-limiting example, the mesh reinforcing material in a foam-basedbiocompatible scaffold of the present invention can be designed to bestiff in one direction, yet elastic in another, or alternatively, themesh reinforcing material can be made isotropic.

During the lyophilization of the reinforced foam, several parameters andprocedures are important to produce implants with the desired integrityand mechanical properties. Preferably, the reinforcement material issubstantially flat when placed in the mold. To ensure the proper degreeof flatness, the reinforcement (e.g., mesh) is pressed flat using aheated press prior to its placement within the mold. Further, in theevent that reinforcing structures are not isotropic it is desirable toindicate this anisotropy by marking the construct to indicatedirectionality. This can be accomplished by embedding one or moreindicators, such as dyed markings or dyed threads, within the wovenreinforcements. The direction or orientation of the indicator willindicate to a surgeon the dimension of the implant in which physicalproperties are superior.

As noted above, the manner in which the polymer solution is added to themold prior to lyophilization helps contribute to the creation of atissue implant with adequate mechanical integrity. Assuming that a meshreinforcing material will be used, and that it will be positionedbetween two thin (e.g., 0.75 mm) shims it should be positioned in asubstantially flat orientation at a desired depth in the mold. Thepolymer solution is poured in a way that allows air bubbles to escapefrom between the layers of the foam component. Preferably, the mold istilted at a desired angle and pouring is effected at a controlled rateto best prevent bubble formation. One of ordinary skill in the art willappreciate that a number of variables will control the tilt angle andpour rate. Generally, the mold should be tilted at an angle of greaterthan about 1 degree to avoid bubble formation. In addition, the rate ofpouring should be slow enough to enable any air bubbles to escape fromthe mold, rather than to be trapped in the mold.

If a mesh material is used as the reinforcing component, the density ofthe mesh openings is an important factor in the formation of a resultingtissue implant with the desired mechanical properties. A low density, oropen knitted mesh material, is preferred. One preferred material is a90:10 copolymer of glycolide and lactide, sold under the tradenameVICRYL (Ethicon, Inc., Somerville, N.J.). One exemplary low density,open knitted mesh is Knitted VICRYL VKM-M, available from Ethicon, Inc.,Somerville, N.J. Other preferred materials are polydioxanone or 95:5copolymer of lactide and glycolide.

The density or “openness” of a mesh material can be evaluated using adigital photocamera interfaced with a computer. In one evaluation, thedensity of the mesh was determined using a Nikon SMZ-U Zoom with a Sonydigital photocamera DKC-5000 interfaced with an IBM 300PL computer.Digital images of sections of each mesh magnified to 20× weremanipulated using Image-Pro Plus 4.0 software in order to determine themesh density. Once a digital image was captured by the software, theimage was thresholded such that the area accounting for the empty spacesin the mesh could be subtracted from the total area of the image. Themesh density was taken to be the percentage of the remaining digitalimage. Implants with the most desirable mechanical properties were foundto be those with a mesh density in the range of about 12 to 80% and morepreferably about 45 to 80%.

In one embodiment, the preferred scaffold for cartilage repair is a meshreinforced foam. More preferably, the foam is reinforced with a meshthat includes polydioxanone (PDO) and the foam composition is acopolymer of 35:65 ε-caprolactone and glycolide. For articularcartilage, the preferred structure to allow cell and tissue ingrowth isone that has an open pore structure and is sized to sufficiently allowcell migration. A suitable pore size is one in which an average diameteris in the range of about 50 to 1000 microns, and more preferably,between about 50 to 500 microns. The mesh layer has a thickness in therange of about 1 micron to 1000 microns. Preferably, the foam has athickness in the range of about 300 microns to 2 mm, and morepreferably, between about 500 microns and 1.5 mm. Preferably, the meshlayer has a mesh density in the range of about 12 to 80% and morepreferably about 45 to 80%.

In another embodiment, the preferred scaffold for cartilage repair is anonwoven structure. More preferably, the composition of the nonwovenstructure are PANACRYL, a 95:5 copolymer of lactide and glycolide,VICRYL, a 90:10 copolymer of glycolide and lactide, or a blend ofpolydioxanone and VICRYL. For articular cartilage, the preferredstructure to allow cell and tissue ingrowth is one that has an open porestructure and is sized to sufficiently allow cell migration. A suitablepore size for the nonwoven scaffold is one in which an average diameteris in the range of about 50 to 1000 microns and more preferably betweenabout 100 to 500 microns. The nonwoven scaffold has a thickness betweenabout 300 microns and 2 mm, and more preferably, between about 500microns and 1.5 mm.

In yet another embodiment, the preferred scaffold for meniscus repair isa mesh reinforced foam. More preferably, the foam is reinforced foamwith a mesh that includes polydioxanone (PDO) and the foam compositionis a copolymer of 35:65 ε-caprolactone and glycolide. The preferredstructure to allow cell and tissue ingrowth is one that has an open porestructure and is sized to sufficiently allow cell migration. A suitablepore size is one in which an average diameter is in the range of about50 to 1000 microns, and more preferably, between about 50 to 500microns. The mesh layer has a thickness in the range of about 1 micronto 1000 microns. Preferably, the foam has a thickness in the range ofabout 300 microns to 2 mm, and more preferably, between about 500microns and 1.5 mm. In this embodiment, the preferred method of use isto surround the minced cartilage tissue with this scaffold material.Preferably, the mesh layer has a mesh density in the range of about 12to 80% and more preferably about 45 to 80%.

In still yet another embodiment, the preferred scaffold for tissuerepair, including cartilage, meniscus, tendon, ligament, and skinrepair, is constructed from a naturally occurring extracellular matrixmaterial (“ECM”), such as that found in the stomach, bladder,alimentary, respiratory, urinary, integumentary, genital tracts, orliver basement membrane of animals. Preferably, the ECM is derived fromthe alimentary tract of mammals, such as cows, sheeps, dogs, cats, andmost preferably from the intestinal tract of pigs. The ECM is preferablysmall intestine submucosa (“SIS”), which can include the tunicasubmucosa, along with basilar portions of the tunica mucosa,particularly the lamina muscularis mucosa and the stratum compactum.

For the purposes of this invention, it is within the definition of anaturally occurring ECM to clean and/or comminute the ECM, or even tocross-link the collagen fibers within the ECM. However, it is not withinthe definition of a naturally occurring ECM to extract and purify thenatural fibers and reform a matrix material from purified naturalfibers. Also, while reference is made to SIS, it is understood thatother naturally occurring ECMs are within the scope of this invention.Thus, as used herein, the terms “naturally occurring extracellularmatrix” or “naturally occurring ECM” are intended to refer toextracellular matrix material that has been cleaned, disinfected,sterilized, and optionally cross-linked.

Where SIS is used, a SIS graft can be harvested in a variety of ways, aswill be understood by one skilled in the art. The resulting graftmaterial can have a variety of geometries and consistencies includingfor example, coiled, helical, spring-like, randomized, branched,sheet-like, tubular, spherical, fragmented, fluidized, comminuted,liquefied, foamed, suspended, gel-like, injectable, powdered, ground,and sheared.

One of ordinary skill in the art will appreciate that the selection of asuitable material for forming the biocompatible scaffold of the presentinvention depends on several factors. These factors include in vivomechanical performance; cell response to the material in terms of cellattachment, proliferation, migration and differentiation;biocompatibility; and optionally, bioabsorption (or biodegradation)kinetics. Other relevant factors include the chemical composition,spatial distribution of the constituents, the molecular weight of thepolymer, and the degree of crystallinity.

A bioactive agent may, optionally, be incorporated within the tissuescaffolds 52 of the present invention. Preferably, the bioactive agentis incorporated within, or coated on, the scaffolds 52 disclosed above.In embodiments where the bioactive agent is coated onto the scaffold 52,the bioactive agent is preferably associated with at least a portion ofthe scaffold 52. The bioactive agents used in the present invention canalso be selected from among a variety of effectors that, when present atthe site of injury, promote healing and/or regeneration of the affectedtissue. In addition to being compounds or agents that actually promoteor expedite healing, the effectors may also include compounds or agentsthat prevent infection (e.g., antimicrobial agents and antibiotics),compounds or agents that reduce inflammation (e.g., anti-inflammatoryagents), compounds that prevent or minimize adhesion formation, such asoxidized regenerated cellulose (e.g., INTERCEED and Surgicel®, availablefrom Ethicon, Inc.), hyaluronic acid, and compounds or agents thatsuppress the immune system (e.g., immunosuppressants).

By way of example, other types of effectors present within the implantof the present invention can include heterologous or autologous growthfactors, proteins (including matrix proteins), peptides, antibodies,enzymes, platelets, platelet rich plasma, glycoproteins, hormones,cytokines, glycosaminoglycans, nucleic acids, analgesics, viruses, virusparticles, and cell types. It is understood that one or more effectorsof the same or different functionality may be incorporated within theimplant.

Examples of suitable effectors include the multitude of heterologous orautologous growth factors known to promote healing and/or regenerationof injured or damaged tissue. These growth factors can be incorporateddirectly into the scaffold, or alternatively, the scaffold can include asource of growth factors, such as for example, platelets. “Bioactiveagents,” as used herein, include one or more of the following:chemotactic agents; therapeutic agents (e.g., antibiotics, steroidal andnon-steroidal analgesics and anti-inflammatories, anti-rejection agentssuch as immunosuppressants and anti-cancer drugs); various proteins(e.g., short term peptides, bone morphogenic proteins, glycoprotein andlipoprotein); cell attachment mediators; biologically active ligands;integrin binding sequence; ligands; various growth and/ordifferentiation agents and fragments thereof (e.g., epidermal growthfactor (EGF), hepatocyte growth factor (HGF), vascular endothelialgrowth factors (VEGF), fibroblast growth factors (e.g., bFGF), plateletderived growth factors (PDGF), insulin derived growth factor (e.g.,IGF-1, IGF-II) and transforming growth factors (e.g., TGF-β I-III),parathyroid hormone, parathyroid hormone related peptide, bonemorphogenetic proteins (e.g., BMP-2, BMP-4; BMP-6; BMP-12), sonichedgehog, growth differentiation factors (e.g., GDF5, GDF6, GDF8),recombinant human growth factors (e.g., MP52), cartilage-derivedmorphogenetic proteins, (CDMP1)); small molecules that affect theupregulation of specific growth factors; tenascin-C; hyaluronic acid;chondroitin sulfate; fibronectin; decorin; thromboelastin;thrombin-derived peptides; heparin-binding domains; heparin; heparansulfate; DNA fragments and DNA plasmids. Suitable effectors likewiseinclude the agonists and antagonists of the agents described above. Thegrowth factor can also include combinations of the growth factorsdescribed above. In addition, the growth factor can be autologous growthfactor that is supplied by platelets in the blood. In this case, thegrowth factor from platelets will be an undefined cocktail of variousgrowth factors. If other such substances have therapeutic value in theorthopaedic field, it is anticipated that at least some of thesesubstances will have use in the present invention, and such substancesshould be included in the meaning of “bioactive agent” and “bioactiveagents” unless expressly limited otherwise.

Biologically derived agents, suitable for use as effectors, include oneor more of the following: bone (autograft, allograft, and xenograft) andderivates of bone; cartilage (autograft, allograft and xenograft),including, for example, meniscal tissue, and derivatives; ligament(autograft, allograft and xenograft) and derivatives; derivatives ofintestinal tissue (autograft, allograft and xenograft), including forexample submucosa; derivatives of stomach tissue (autograft, allograftand xenograft), including for example submucosa; derivatives of bladdertissue (autograft, allograft and xenograft), including for examplesubmucosa; derivatives of alimentary tissue (autograft, allograft andxenograft), including for example submucosa; derivatives of respiratorytissue (autograft, allograft and xenograft), including for examplesubmucosa; derivatives of genital tissue (autograft, allograft andxenograft), including for example submucosa; derivatives of liver tissue(autograft, allograft and xenograft), including for example liverbasement membrane; derivatives of skin tissue; platelet rich plasma(PRP), platelet poor plasma, bone marrow aspirate, demineralized bonematrix, insulin derived growth factor, whole blood, fibrin and bloodclot. Purified ECM and other collagen sources are also appropriatebiologically derived agents. If other such substances have therapeuticvalue in the orthopaedic field, it is anticipated that at least some ofthese substances will have use in the present invention, and suchsubstances should be included in the meaning of “biologically derivedagent” and “biologically derived agents” unless expressly limitedotherwise.

Biologically derived agents also include bioremodelable collageneoustissue matrices. The terms “bioremodelable collagenous tissue matrix”and “naturally occurring bioremodelable collageneous tissue matrix”include matrices derived from native tissue selected from the groupconsisting of skin, artery, vein, pericardium, heart valve, dura mater,ligament, bone, cartilage, bladder, liver, stomach, fascia andintestine, whatever the source. Although the term “naturally occurringbioremodelable collageneous tissue matrix” is intended to refer tomatrix material that has been cleaned, processed, sterilized, andoptionally crosslinked, it is not within the definition of a naturallyoccurring bioremodelable collageneous tissue matrix to purify thenatural fibers and reform a matrix material from purified naturalfibers.

The proteins that may be present within the implant include proteinsthat are secreted from a cell or other biological source, such as forexample, a platelet, which is housed within the implant, as well asthose that are present within the implant in an isolated form. Theisolated form of a protein typically is one that is about 55% or greaterin purity, i.e., isolated from other cellular proteins, molecules,debris, etc. More preferably, the isolated protein is one that is atleast 65% pure, and most preferably one that is at least about 75 to 95%pure. Notwithstanding the above, one of ordinary skill in the art willappreciate that proteins having a purity below about 55% are stillconsidered to be within the scope of this invention. As used herein, theterm “protein” embraces glycoproteins, lipoproteins, proteoglycans,peptides, and fragments thereof. Examples of proteins useful aseffectors include, but are not limited to, pleiotrophin, endothelin,tenascin, fibronectin, fibrinogen, vitronectin, V-CAM, I-CAM, N-CAM,selectin, cadherin, integrin, laminin, actin, myosin, collagen,microfilament, intermediate filament, antibody, elastin, fibrillin, andfragments thereof.

Glycosaminoglycans, highly charged polysaccharides which play a role incellular adhesion, may also serve as effectors according to the presentinvention. Exemplary glycosaminoglycans useful as effectors include, butare not limited to, heparan sulfate, heparin, chondroitin sulfate,dermatan sulfate, keratan sulfate, hyaluronan (also known as hyaluronicacid), and combinations thereof.

The tissue scaffolds 52 of the present invention can also have cellsincorporated therein. Suitable cell types that can serve as effectorsaccording to this invention include, but are not limited to, osteocytes,osteoblasts, osteoclasts, fibroblasts, stem cells, pluripotent cells,chondrocyte progenitors, chondrocytes, endothelial cells, macrophages,leukocytes, adipocytes, monocytes, plasma cells, mast cells, umbilicalcord cells, stromal cells, mesenchymal stem cells, epithelial cells,myoblasts, tenocytes, ligament fibroblasts, neurons, bone marrow cells,synoviocytes, embryonic stem cells; precursor cells derived from adiposetissue; peripheral blood progenitor cells; stem cells isolated fromadult tissue; genetically transformed cells; a combination ofchondrocytes and other cells; a combination of osteocytes and othercells; a combination of synoviocytes and other cells; a combination ofbone marrow cells and other cells; a combination of mesenchymal cellsand other cells; a combination of stromal cells and other cells; acombination of stem cells and other cells; a combination of embryonicstem cells and other cells; a combination of precursor cells isolatedfrom adult tissue and other cells; a combination of peripheral bloodprogenitor cells and other cells; a combination of stem cells isolatedfrom adult tissue and other cells; and a combination of geneticallytransformed cells and other cells. If other cells are found to havetherapeutic value in the orthopaedic field, it is anticipated that atleast some of these cells will have use in the present invention, andsuch cells should be included within the meaning of “cell” and “cells”unless expressly limited.

Cells typically have at their surface receptor molecules which areresponsive to a cognate ligand (e.g., a stimulator). A stimulator is aligand which when in contact with its cognate receptor induce the cellpossessing the receptor to produce a specific biological action. Forexample, in response to a stimulator (or ligand) a cell may producesignificant levels of secondary messengers, like Ca⁺², which then willhave subsequent effects upon cellular processes such as thephosphorylation of proteins, such as (keeping with our example) proteinkinase C. In some instances, once a cell is stimulated with the properstimulator, the cell secretes a cellular messenger usually in the formof a protein (including glycoproteins, proteoglycans, and lipoproteins).This cellular messenger can be an antibody (e.g., secreted from plasmacells), a hormone, (e.g., a paracrine, autocrine, or exocrine hormone),a cytokine, or natural or synthetic fragments thereof.

The tissue implants 50 of the invention can also be used in gene therapytechniques in which nucleic acids, viruses, or virus particles deliver agene of interest, which encodes at least one gene product of interest,to specific cells or cell types. Accordingly, the biological effectorcan be a nucleic acid (e.g., DNA, RNA, or an oligonucleotide), a virus,a virus particle, or a non-viral vector. The viruses and virus particlesmay be, or may be derived from, DNA or RNA viruses. The gene product ofinterest is preferably selected from the group consisting of proteins,polypeptides, interference ribonucleic acids (iRNA) and combinationsthereof.

Once the applicable nucleic acids and/or viral agents (i.e., viruses orviral particles) are incorporated into the biocompatible scaffold of thetissue repair implant, the implant can then be implanted into aparticular site to elicit a type of biological response. The nucleicacid or viral agent can then be taken up by the cells and any proteinsthat they encode can be produced locally by the cells. In oneembodiment, the nucleic acid or viral agent can be taken up by the cellswithin the tissue fragment of the minced tissue suspension, or, in analternative embodiment, the nucleic acid or viral agent can be taken upby the cells in the tissue surrounding the site of the injured tissue.One skilled in the art will recognize that the protein produced can be aprotein of the type noted above, or a similar protein that facilitatesan enhanced capacity of the tissue to heal an injury or a disease,combat an infection, or reduce an inflammatory response. Nucleic acidscan also be used to block the expression of unwanted gene product thatmay impact negatively on a tissue repair process or other normalbiological processes. DNA, RNA and viral agents are often used toaccomplish such an expression blocking function, which is also known asgene expression knock out.

One of ordinary skill in the art will appreciate that the identity ofthe bioactive agent may be determined by a surgeon, based on principlesof medical science and the applicable treatment objectives. It isunderstood that the bioactive agent or effector of the issue repairimplant can be incorporated within the tissue scaffold 52 before orafter manufacture of the tissue scaffold 52, or before or after thesurgical placement of the implant 50.

Prior to surgical placement, the tissue scaffold 52 can be placed in asuitable container comprising the bioactive agent. After an appropriatetime and under suitable conditions, the scaffold 52 will becomeimpregnated with the bioactive agent. Alternatively, the bioactive agentcan be incorporated within the scaffold 52 by, for example, using anappropriately gauged syringe to inject the biological agent(s) into thescaffold. Other methods well known to those of skilled in the art can beapplied in order to load a scaffold 52 with an appropriate bioactiveagent, such as mixing, pressing, spreading, centrifuging and placing thebioactive agent into the scaffold 52. Alternatively, the bioactive agentcan be mixed with a gel-like carrier prior to injection into thescaffold 52.

Following surgical placement, an implant wherein the biocompatiblescaffold 52 is devoid of any bioactive agent can be infused withbiological agent(s), or an implant wherein the scaffold includes atleast one bioactive agent can be augmented with a supplemental quantityof the bioactive agent. One method of incorporating a bioactive agentwithin a surgically installed implant is by injection using anappropriately gauged syringe.

The amount of the bioactive agent included with a biocompatible scaffold52 will vary depending on a variety of factors, including the size ofthe scaffold, the material from which the scaffold is made, the porosityof the scaffold, the identity of the biologically component, and theintended purpose of the tissue repair implant. One skilled in the artcan readily determine the appropriate quantity of bioactive agent toinclude within a biocompatible scaffold for a given application in orderto facilitate and/or expedite the healing of tissue. The amount ofbioactive agent will, of course, vary depending upon the identity of thebioactive agent and the given application.

The tissue repair implants of the present invention can be used in avariety of surgical and non-surgical applications. In some surgicalapplications, such as for use in the repair of a variety of tissuesincluding a torn ligament, tendon, rotator cuff, nerve, skin, cartilage,or meniscus, the tissue implants of the invention must be able to behandled in the operating room, and they must be able to be sutured orotherwise fastened without tearing. Additionally, the implants shouldhave a structure suitable to encourage tissue ingrowth.

In one embodiment of the present invention, the tissue repair implant isused in the treatment of a tissue injury, such as injury to a ligament,tendon, nerve, skin, cartilage or meniscus. Repairing tissue injuriesinvolves the steps of obtaining a slice of living tissue 22 by any ofthe variety of techniques known to those skilled in the art, and placingthe tissue slice 22 in a desired position relative to the tissue injury.While a single tissue slice 22 can be used, more than one tissue slice22 can be joined together to form a layered implant 40 for implantation.Repairing tissue injuries may also involve depositing the tissue slice22 onto a biocompatible, bioabsorbable tissue scaffold 52 such that thetissue slice 22 becomes associated with the scaffold 52 to form a tissuerepair implant 50. A retaining element 30 can optionally be applied tosecure the implant to the injury or defect 10. In an additional step,finely minced tissue fragments can be applied to the implant to enhancethe effectiveness of the regrowth and healing process. The cells in boththe tissue slices and minced tissue fragments can migrate out and beginproliferating and integrating with surrounding tissue at the site ofimplantation, thereby repairing the tissue injury. This method forrepairing tissue injuries can include an additional, optional step.Prior to the step of placing the tissue repair implant in a desiredposition relative to the tissue injury, the scaffold and associatedtissue particles can be incubated for a duration and under conditionseffective to allow cells within the tissue particles to migrate from thetissue and begin populating the scaffold.

The implants used to repair injured tissue can be of a size and shapesuch that they match the geometry and dimensions of a desired portion orlesion of the tissue to be treated. The implant can be sized and shapedto produce the necessary geometry by numerous techniques includingcutting, folding, rolling, or otherwise manipulating the implant. Asnoted above, the bioactive agent may be added to the scaffold during orafter manufacture of the scaffold or before or after the implant isinstalled in a patient. An additional quantity of the bioactive agentmay be added after the implant is installed. Once access is made intothe affected anatomical site (whether by minimally invasive, open ormini-open surgical technique), the implant can be affixed to a desiredposition relative to the tissue injury, such as within a tear or lesion.Once the implant is placed in the desired position or lesion, it can beaffixed by using an appropriate retaining element 30 or other suitabletechnique. In one aspect, the implant can be affixed by a chemicaland/or mechanical fastening technique. Suitable chemical fastenersinclude glues and/or adhesive such as fibrin glue, fibrin clot, andother known biologically compatible adhesives. Suitable mechanicalfasteners include sutures, staples, tissue tacks, suture anchors, darts,screws, pins and arrows. It is understood that combinations of one ormore chemical and/or mechanical fasteners can be used. Alternatively,one need not use any chemical and/or mechanical fasteners. Instead,placement of the implant can be accomplished through an interference fitof the implant with an appropriate site in the tissue to be treated.

In one use, the tissue repair implant can be for repair and to augmenttissue loss during tendon or ligament repair surgery or it can be usedas a stand alone device. In the case of repair, tendon or ligament endsare approximated through appropriate surgical techniques and the tissuerepair implant is used around the joined end to give more mechanicalsupport and to enhance the healing response. As a result of the healingprocess, the tendon or ligament tissue grows within the implant device,eventually maturing into a tissue with similar mechanical properties tothat of native tissue. The implant provides the mechanical support thatis initially necessary to ensure proper healing, and it also serves as aguide for tissue regeneration. In another use as a stand alone device,the ruptured tissue is removed, and the tissue repair implant withsliced tissue serves to replace the function of the damaged tissue. Inone embodiment, the ruptured tissue can be the tissue source used forhealing damaged tissue.

In embodiments where the tissue repair implant is used to repairligament tissue, the tissue repair implant can be used for tissueaugmentation, or alternatively, as a stand-alone device. In embodimentswhere the tissue repair implant is used for augmentation, the tissuerepair implant can be used in conjunction with any of a variety ofstandard, established repair techniques known to those skilled in theart. In embodiments where the tissue repair implant is used foraugmentation during ACL repair, surgeons currently use an autograftconsisting of ligament tissue, bone-patellar tendons, tendon-bonetendons, hamstring tendons, or iliotibial band to repair tissue, and thetissue repair implant of the present invention can be placed eitheraround the autograft, surrounded by the autograft, or alongside theautograft. In embodiments where the tissue repair element is used as astand-alone device, the ruptured ligament can be removed and completelyreplaced by the tissue repair implant. In this case, the tissue repairimplant can be affixed to bone at each end of the implant. In the caseof ACL repair, one end of the implant can be stabilized at the originalorigin site of the femur, while the other end can be placed at theoriginal insertion site on the tibia.

The tissue repair implant can be utilized in a variety ofconfigurations. For example, the implant can be composed of long piecesof tissue, folded or stacked in multiple laminates, or it can be rolledinto the shape or a tube-like structure. Tendon or ligament ends can bejoined, for example, by suturing, stapling, clipping, adhering, oranchoring, the implant to ends of the implant. In some embodiments wherethe tissue repair implant is used to repair tendons, such as forexample, rotator cuff repair, the surgeon can use the tissue repairimplant to assist in the reapproximation of the tom rotator cuff to abony trough through the cortical surface of the greater tuberosity.Often times, in older patients, the rotator cuff tissue is thin anddegenerate and/or the quality of the humerus is osteoporotic. Therefore,in order to increase the strength of the attachment to the bony trough,the tissue repair implant can be placed on top of the tendon, such thatthe sutures would pass through both the scaffold and tendon, oralternatively, the tissue repair implant can be used on top of the bonebridge to prevent the sutures from pulling out of the bone. In eitherembodiment, the tissue repair implant provides suture retentionstrength. In situations where the quality of the rotator cuff is sodegenerate that the tissue cannot be reapproximated to the humerus, thetissue repair implant can serve as a bridge, wherein one end of theimplant can be joined to the remaining tendon while the other end can beattached to the bone.

In another variation, the implant can be used to repair or replace thesheath of a tendon. To do so, the implant is sutured or otherwise joinedto the connective tissue, such as the periosteum, synovium, or muscle,or wrapped around the tendon. This construction allows free gliding ofthe tendon within the sheath formed by the implant. The implant providesthe necessary structural support following surgery. Over time, however,the implant in this embodiment can be resorbed and replaced by newtissue.

The implants of the invention can also be used for organ repairreplacement or regeneration strategies that may benefit from theseunique tissue implants. For example, these implants can be used forspinal disc, cranial tissue, dura, nerve tissue, liver, pancreas,kidney, bladder, uterus, esophagus, liver spleen, cardiac muscle,skeletal muscle, skin, fascia, pelvic floor, stomach, tendons,cartilage, ligaments, and breast tissues.

The implants of the present invention can also be used as a deliverydevice for a therapeutic, wherein the therapeutic is the minced tissue,which includes a combination of cells, extracellular matrix and inherentgrowth factors. The scaffold portion of the implant can allow forhormones and proteins to be released into the surrounding environment.

The methods of repairing tissue injuries using the tissue implantsaccording to the present invention can be conducted during a surgicaloperation to repair the tissue injury. In an exemplary method, a patientis prepared for tissue repair surgery in a conventional manner usingconventional surgical techniques. Tissue repair is performed at the siteof the defective or injured tissue 10 using the composite tissue implant50 of the present invention. The tissue slice 22 used to form the tissueimplant 50 is obtained from the patient (or another donor) usingappropriate tools and techniques. The tissue slice 22 is eitherharvested with a specified geometry suitable for the defect or injury 10or cut into the specified geometry after harvest. The method ofharvesting or cutting into the specified geometry can be done with aconventional sterile surgical instruments or a specially designeddevice. The prepared tissue slice is then applied to a tissue scaffold52. The scaffold and tissue can then be implanted at the site of tissueinjury using a retaining element 30 such as sutures, staples, anadhesive agent, mechanical force or any other fixation device. Finalwound closure is performed in a conventional manner using conventionalsurgical techniques.

The following examples are illustrative of the principles and practiceof this invention. Numerous additional embodiments within the scope andspirit of the invention will become apparent to those skilled in theart.

EXAMPLE 1

In this in vitro study, cellular migration and new matrix formation fromminced and shredded bovine anterior cruciate ligament (ACL) tissue intonon-woven tissue scaffold (PANACRYL) was evaluated and compared.Pre-scored and sterilized PANACRYL non-woven sheets were trimmed toyield two (2) 2.5×2 cm sheets. Next, bovine ACL tissue samples wereobtained from two knees from the same animal. To prepare the shreddedtissue, an isolated section of the bovine ACL was trimmed under asepticconditions to measure approximately 2×2×0.5 cm in overall dimensions.Using a sterile scalpel, multiple full thick incisions were madeparallel to the fibers of the ACL section, yielding tissue strandsmeasuring approximately 2 cm in length and 0.1 cm in maximum diameter.The tissue strands were placed parallel to the long axis of a PANACRYLsheet to form a composite implant. To prepare the control, minced ACLtissue fragments were also applied to a sheet of PANACRYL. The mincedtissue fragments were obtained by mincing the bovine ACL tissue sampleusing scalpel blades to obtain small tissue fragments. Both thecomposite implant and the control were placed in Dulbecco's modifiedeagles medium (DMEM), supplemented with 20% fetal bovine serum (FBS).After 4 days and 21 days, a DNA assay was performed and the histology ofthe samples were evaluated.

Results

After 4 days and 21 days, the samples were prepared for histologicalevaluation. Five-micron sections were obtained and adhered to gluecoated slides. These sections were then stained with hematoxylin andeosin. In addition, the DNA content of each sample was obtained by assayusing a Molecular Probes CyQUANT Cell Proliferation Assay kit (cat. no.C-7026). 5 mm bunch biopsy samples of the composite implant wereobtained at day 4 and day 21. The samples were washed once in 1×PBS andfrozen at −20° C. for at least one hour. The samples were then thawed atroom temperature and incubated in 40 μl of 4M Guanidine-HCL. 10 μl ofthe guanidine digested sample was added to 190 μl of CyQUANT GR workingsolution. The mixture was vortexed and incubated for 5 minutes, and thenloaded into a 96-well black walled plate and analyzed byspectrophotometry. The results of the DNA assay are shown in Table 1below.

In the control sample with the minced tissue, the cells within theminced tissue appeared viable after 4 days, while no cells were notedwithin the tissue scaffold. After 21 days, an evenly distributed sparsecell population was noted within the scaffold. The foci of what appearedto be early new matrix formation was noted along the tissue-scaffoldjunction.

In the shredded tissue implant, the cells within the shredded tissueappeared viable after 4 days, while no cells were observed within thetissue scaffold. After 21 days, an evenly distributed sparse cellpopulation was noted within the scaffold. The foci of what appeared tobe early new matrix formation was noted along the tissue-scaffoldjunction. TABLE 1 Comparison of DNA content in minced v. shredded tissueDNA assay: DNA (ng) sample day 4 day 4 avg. day 21 day 21 avg. Minced4420 4284 5256 4853.66667 4198 3862 4234 5443 Shredded 1793 2033.3332400 2680.66667 2074 2219 2233 3423

The data from Table 1 is also graphically presented in FIG. 5 as a barchart for ease of comparison.

Discussion

As indicated by the histological evaluation of the samples, the cells ofthe shredded ACL tissue were able to migrate into the tissue scaffoldand show early signs of matrix formation at 21 days. Shredded ACL tissuealso appeared to function similarly to minced ACL tissue fragments inthat both tissue geometries exhibited the same cell population anddistribution profile at 21 days.

As indicated by the DNA assays performed, the relative increase in DNAcontent noted in the shredded ACL tissue appears similar to the increasein DNA content noted in the minced ACL tissue. These results areconsistent with the histological data.

It was concluded that sparse and evenly distributed cell migration andfocal new matrix formation can be observed in PANACRYL non-wovenscaffolds seeded with shredded bovine ACL tissue at 21 days. Theseresults are similar to minced bovine ACL tissue fragments seeded ontothe same scaffold at 21 days.

EXAMPLE 2

In this in vitro study, sliced meniscal tissue was tested as a source ofviable cells for meniscal regeneration. First, an isolated bovinemeniscus was obtained and trimmed to remove the surrounding synovium.Using a sterile dermatome, slices of meniscus were removed. Thethickness of the slices were either 200 μm, 300 μm or 500 μm. The sliceswere approximately 1 to 2 cm in length. The meniscal slices were seededonto scaffolds comprising sterilized, 65:35 polyglycolic acid/polycaprolactone acide foam reinforced with polydioxone mesh at a density of20 mg/cm². The scaffolds measured 4×2.5 cm. Platelet rich plasma (PRP)was added to the scaffolds at a concentration of 20 μl/cm² and thescaffolds cultured for 3 and 5 weeks in Dulbecco's modified eaglesmedium (DMEM) supplemented with 0.5% fetal bovine serum (FBS). After 3and 5 weeks, the samples were prepared for histological evaluation.Sections of the samples were obtained and stained with hematoxylin andeosin.

Results

FIGS. 6A and 6B demonstrate migration of viable fibrochondrocytes fromtissue slices of 200 μm (FIG. 6A) and 300 μm thickness (FIG. 6B), at 3weeks. FIG. 6C shows similar cell migration from 500 μm thick tissue at3 weeks. At 5 weeks, similar cell migration patterns were observed foreach of the varying tissue slices.

Discussion

The study shows that cells in the sliced meniscal tissue were viable andable to migrate into and populate tissue scaffolds associated with thesliced tissue. In addition, the variation in thickness of the slices didnot appear to have a qualitative difference in the cell population inthe scaffolds.

EXAMPLE 3

In this in vitro study, minced tissue fragments were used in conjunctionwith mosaicplasty techniques to demonstrate that better integrationbetween cartilage plugs can be achieved and cartilage repair of damagedtissue can be enhanced by the addition of minced cartilage fragments.

Healthy articular cartilage was obtained from bovine stifle. A 3 mmbiopsy punch was used to punch cylinders or plugs of cartilage tissue.The rest of the cartilage tissue, which was substantially free of bone,was minced using scalpel blades to obtain small tissue fragments. Thesize of the tissue fragments varied but was less than or equal to 1×1 mmin dimension. Four 3 mm cartilage cylinders were placed together inparallel to each other longitudinally in a glass cylinder with an innerdiameter of 8 mm. In one group, a blood clot was then formed inside theglass cylinder to keep the tissues together. In another group, theminced cartilage tissue was placed in the glass cylinder with four 3 mmcartilage cylinders and then a blood clot was formed inside to keepeverything together. The glass cylinders were slipped off and thetissue-clot was placed in culture in 6 well plates containingchondrocyte growth medium. The chondrocyte growth medium consisted ofDulbecco's modified eagles medium (DMEM-high glucose) supplemented with10% fetal calf serum (FCS), 10 mM HEPES, 0.1 mM nonessential aminoacids, 20 mg/ml of L-proline, 50 mg/ml ascorbic acid, 100 mg/mlpenicillin, 100 mg/ml of streptomycin. The growth medium was changedevery other day. The tissues were cultured at 37° C. in a cell cultureincubator for six weeks. Samples were removed, macroscopic pictures weretaken, and then the samples were placed in formalin for histology.Sections were stained with H&E and Safranin-O. FIG. 7A is a photographof the group with minced tissue which shows that all the tissues areheld together. Histology of this sample confirmed that cells from boththe minced tissue and cylinders were migrating into the space betweenthe tissue cylinders, keeping the whole entity together (FIG. 7C). FIG.7B is a photograph of the group without the minced tissue, showing thatafter 3 weeks in culture the cartilage cylinders began pulling away fromeach other because there was nothing that was bonding them together.

Discussion

This study shows that the addition of minced cartilage fragments toclosely associated cartilage plugs or cylinders can produce bettercellular integration between the plugs. While cartilage cylinders wereused in the present example, it is contemplated that the samemosaicplasty principles can be applied to the present invention toprovide a tissue repair implant comprising tissue slices and mincedtissue fragments for enhanced cellular integration and tissue repair.

One of ordinary skill in the art will appreciate further features andadvantages of the invention based on the above-described embodiments.Accordingly, the invention is not to be limited by what has beenparticularly shown and described, except as indicated by the appendedclaims. All publications and references cited herein are expresslyincorporated herein by reference in their entirety.

1. A biocompatible tissue implant for repairing a tissue injury ordefect, comprising: a biological tissue slice having a geometry suitablefor implantation at the tissue site, the tissue slice including aneffective amount of viable cells, and further being dimensioned so thatthe cells can migrate out of the tissue slice to proliferate andintegrate with tissue at the injury or defect.
 2. The implant of claim1, wherein the tissue slice comprises autogeneic tissue, allogeneictissue, xenogeneic tissue, and combinations thereof.
 3. The implant ofclaim 1, wherein the tissue slice is obtained from a tissue typeselected from the group consisting of cartilage, meniscus, tendon,ligament, intestinal, stomach, bladder, alimentary, respiratory,genital, liver, dermis, synovium, and combinations thereof.
 4. Theimplant of claim 1, wherein the tissue slice has a thickness less thanabout 3 mm.
 5. The implant of claim 4, wherein the tissue slice has athickness less than about 1 mm.
 6. The implant of claim 5, wherein thetissue slice has a thickness in the range of about 200 μm to about 500μm.
 7. The implant of claim 1, further including a plurality of tissueslices joined together to form a layered implant of a desired size andgeometry.
 8. The implant of claim 1, further including a retainingelement for securing the tissue slice to the tissue site.
 9. The implantof claim 8, wherein the retaining element is selected from the groupconsisting of fasteners, staples, tissue tacks, sutures, adhesives, andcombinations thereof.
 10. The implant of claim 9, wherein the retainingelement is an adhesive selected from the group consisting of selectedfrom the group consisting of hyaluronic acid, fibrin glue, fibrin clot,collagen gel, collagen-based adhesive, alginate gel, crosslinkedalginate, gelatin-resorcin-formalin-based adhesive, mussel-basedadhesive, dihydroxyphenylalanine (DOPA)-based adhesive, chitosan,transglutaminase, poly(amino acid)-based adhesive, cellulose-basedadhesive, polysaccharide-based adhesive, synthetic acrylate-basedadhesives, platelet rich plasma (PRP), platelet poor plasma (PPP), PRPclot, PPP clot, blood, blood clot, polyethylene glycol-based adhesive,Matrigel, Monostearoyl Glycerol co-Succinate (MGSA), MonostearoylGlycerol co-Succinate/polyethylene glycol (MGSA/PEG) copolymers,laminin, elastin, proteoglycans, and combinations thereof.
 11. Theimplant of claim 1, further including at least one minced tissuefragment containing a plurality of viable cells.
 12. The implant ofclaim 11, wherein the at least one minced tissue fragment is deliveredin a biological or synthetic hydrogel selected from the group consistingof hyaluronic acid, fibrin glue, fibrin clot, collagen gel,collagen-based adhesive, alginate gel, crosslinked alginate, chitosan,synthetic acrylate-based gels, platelet rich plasma (PRP), platelet poorplasma (PPP), PRP clot, PPP clot, blood, blood clot, Matrigel, agarose,chitin, chitosan, polysaccharides, poly(oxyalkylene), a copolymer ofpoly(ethylene oxide)-poly(propylene oxide), poly(vinyl alcohol),laminin, elastin, proteoglycans, solubilized basement membrane, orcombinations thereof.
 13. The implant of claim 11, wherein the at leastone minced tissue fragment has a particle size in the range of about 0.1mm³ to about 2 mm³.
 14. The implant of claim 1, further including abiocompatible tissue scaffold.
 15. The implant of claim 14, wherein thetissue scaffold is bioresorbable.
 16. The implant of claim 14, whereinthe tissue scaffold is formed from a material selected from the groupconsisting of a synthetic polymer, a natural polymer, an injectable gel,a ceramic material, autogeneic tissue, allogeneic tissue, xenogeneictissue, and combinations thereof.
 17. The implant of claim 14, whereinthe scaffold further comprises at least one bioactive agent appliedthereto.
 18. The implant of claim 17, wherein the at least one bioactiveagent is selected from the group consisting of growth factors, matrixproteins, peptides, antibodies, enzymes, platelets, platelet richplasma, glycoproteins, hormones, glycosaminoglycans, nucleic acids,analgesics, viruses, virus particles, cytokines and isolated cells andcombinations thereof.
 19. The implant of claim 14, further including aplurality of tissue slices and a plurality of tissue scaffolds joinedtogether to form a layered implant of a desired size and geometry.
 20. Amethod for repairing a tissue injury or defect, comprising: providing abiocompatible tissue implant comprising a biological tissue slice havinga geometry suitable for implantation at a tissue injury or defect site,the tissue slice including an effective amount of viable cells, andfurther being dimensioned so that the cells can migrate out of thetissue slice to proliferate and integrate with tissue at the tissueinjury or defect site; and delivering the implant to the tissue site tobe repaired.
 21. The method of claim 20, wherein the biocompatibletissue implant is in the form of a plurality of tissue slices joinedtogether to form a layered implant of a desired size and geometry. 22.The method of claim 20, further including the step of applying thetissue slice to a biocompatible tissue scaffold to form a compositeimplant prior to the delivering step, and the step of delivering theimplant comprises delivering the composite implant to the tissue site tobe repaired.
 23. The method of claim 20, further including the step ofapplying a bioactive agent to the implant either before or afterdelivery.
 24. The method of claim 23, wherein the bioactive agent isselected from the group consisting of growth factors, matrix proteins,peptides, antibodies, enzymes, platelets, platelet rich plasma,glycoproteins, hormones, glycosaminoglycans, nucleic acids, analgesics,viruses, virus particles, cytokines and isolated cells and combinationsthereof.
 25. The method of claim 20, further including the step ofsecuring the biocompatible tissue implant to the tissue site using aretaining element selected from the group consisting of fasteners,staples, tissue tacks, sutures, adhesives, and combinations thereof. 26.The method of claim 20, further including the step of applying at leastone minced tissue fragment containing a plurality of viable cells to thetissue implant prior to the delivering step.
 27. The method of claim 26,wherein the at least one minced tissue fragment is applied in abiological or synthetic hydrogel selected from the group consisting ofhyaluronic acid, fibrin glue, fibrin clot, collagen gel, collagen-basedadhesive, alginate gel, crosslinked alginate, chitosan, syntheticacrylate-based gels, platelet rich plasma (PRP), platelet poor plasma(PPP), PRP clot, PPP clot, blood, blood clot, Matrigel, agarose, chitin,chitosan, polysaccharides, poly(oxyalkylene), a copolymer ofpoly(ethylene oxide)-poly(propylene oxide), poly(vinyl alcohol),laminin, elastin, proteoglycans, solubilized basement membrane, orcombinations thereof.